1. Field of the Invention
The present invention generally relates to the transportation, robotic crystal mounting and alignment, manipulation, mounting, alignment of crystal samples in a variety of experimental environments. The present invention more particularly relates to the mounting, alignment, and exposure of samples to synchrotron radiation for high-speed x-ray crystallography.
2. Description of the Relevant Art
Overview of X-ray Crystallographic Systems
Aspects of the present invention facilitate the transportation, as well as the remote and unattended mounting and alignment of frozen crystals—of e.g. biological materials, such as proteins, lipids, or deoxyribonucleic acids (DNA)—for x-ray diffraction analysis. A major challenge in the x-ray diffraction analysis system design is the necessity of storing the samples in liquid nitrogen before mounting and following removal, as well as maintaining the samples at near liquid nitrogen temperature throughout the mounting, alignment, and x-ray diffraction analysis data acquisition process. Additionally, the precision and stability of the crystal sample location alignment must be very high, with absolute sample position maintained within a few microns while rotating in one axis while being exposed to an incident x-ray beam through as much as a full 360°.
Traditional x-ray diffraction analysis crystal sample handling procedures are operator-intensive, requiring continuous manual operator intervention at the measurement station. The ability to perform these tasks remotely and automatically significantly increases crystal mounting and measurement throughput, as well as reliability for large-scale protein crystallography characterization. An increase in throughput multiplies the number of samples that may be analyzed in a given time period, thus decreasing the time per sample, thereby lowering the cost associated with synchrotron-based x-ray crystallography.
Synchrotron-Based X-ray Crystallography
One embodiment of this invention is in the area of cryogenic protein crystallography at synchrotron sources, although the robotic mounting and alignment system can be adapted for other laboratory x-ray sources. Potential uses include high-volume protein characterization experiments. The level of application of this invention could range from a small experimental program processing only a few samples per day to large projects screening and analyzing many thousands of samples per year.
Synchrotron-based x-ray crystallography is one application of this invention. Synchrotrons are capable of producing intense monochromatic pseudo-coherent photons of precisely controllable energies. The property of high intensity (otherwise known as high brightness) of the synchrotron x-ray beams means that acquisition of crystal lattice diffraction patterns can be done very rapidly, whereas other, lower intensity beams may require several times longer for a diffraction pattern to be acquired. The high brightness of the synchrotron radiation, combined with the narrow energy bandwidth achievable using a monochromator, can lead to exceedingly high-resolution x-ray diffraction patterns.
The x-ray diffraction patterns can subsequently be analyzed to infer the relative spatial positions of the atoms constituting the crystal lattice structure. The overall x-ray diffraction analysis of crystals is known as x-ray crystallography. The information contained in the crystal structure can lead to important insights about the function of the molecule and into molecular-chemical interactions. Such insights can lead to targeted, and thus faster, pharmaceutical development and improved pharmaceuticals: a field known as ‘structure based pharmaceutical design’.
Biological Crystallography Mounting Techniques
Currently, most x-ray crystallography work is done using synchrotron x-ray sources. These x-ray sources are extremely expensive to operate, which means that time is precious. However, since synchrotron x-ray crystallography is still a recent phenomenon, most sample mounting is done manually, which is both slow and imprecise. Furthermore, since crystallography must be done on crystalline material, the sample must be maintained in a frozen state. Typically, this is ensured by keeping the sample at near liquid nitrogen temperatures.
The requirement that the sample be maintained at liquid nitrogen temperature, however, requires that technicians and scientists can only mount the samples using cumbersome techniques of indirectly handling the sample. Thus, people cannot be allowed to inadvertently heat the sample, and reciprocally, the sample handling tools, and sample handling fixtures, cannot freeze the fingers of the people who do the mounting. To meet this requirement, clumsy tools resembling forceps or pliers are used. These tools are somewhat cumbersome, further adding time and difficulty in mounting and handling the sample.
As more time is required to manually mount the sample, more heat is transferred from the ambient atmosphere, raising the crystal sample temperature. Some biological crystal samples, frozen at a critical point in a chemical reaction with another compound, continue their reactions at temperatures as low at 100° K., only about 22° K. above that of liquid nitrogen. This stringent maximum temperature requirement for some samples implies that the sample must be actively cooled during the entire mounting process, which adds still further time and complexity to the mounting process. It is preferable that the sample crystals be cooled to a temperature not in excess of 150° K., more preferably not in excess of 130° K., yet more preferably not in excess of 110° K., still more preferably not in excess of 100° K., yet still more preferably not in excess of 90° K., and most preferably not in excess of 80° K.
The largest time-related issue with manual operator mounting of synchrotron x-ray crystallography samples is that the humans must enter the x-ray irradiation area (‘the hutch’) to mount and dismount the crystal samples. This action involves in turn a sequence of safety interlocking steps to protect the personnel from a harmful and potentially lethal dosage of x-rays used to irradiate the crystal sample to generate the diffraction patterns. Typically, one or more heavy lead-lined doors must be opened and closed, additional beam shutters inserted, and interlocking safety devices must be carefully verified for safe operation, prior to human access to the sample.
The result is that manual mounting of a synchrotron x-ray crystallography sample is slow. As a result of being slow, manual mounting is very expensive as measured in synchrotron beam time.
Biological Crystallography Sample Transportation
Currently, there are relatively few synchrotron x-ray sources available for x-ray crystallography. Therefore, scientists wishing to use synchrotron x-ray sources face the dilemma of transporting the crystal samples to the synchrotron while simultaneously maintaining the crystal's cryogenically frozen state.
A particularly fruitful use of synchrotron x-ray crystallography is in detection of chemical interactions within a specific biological sample. These interactions are evanescent in nature, sometimes reacting in the one-nanosecond time scale. Additionally, typical biological processes can follow a number of biochemical pathways that are time dependent. Some of these biochemical pathways proceed even at temperatures as low as 100° K. Thus, for a scientist to determine the crystalline structure of an intermediate state biochemical interaction, the biological sample must be frozen to a temperature low enough to inhibit further reaction, typically close to the liquid nitrogen temperature of about 77° K. under normal laboratory conditions.
A Relevant Patent
Abbott Laboratories is the named assignee of U.S. Pat. No. 6,404,849 B1 (the '849 patent), entitled “Automated Sample Handling for X-Ray Crystallography”. The '849 patent discloses algorithms for centering a crystal at a reference position relative to home position sensors, as well as the hardware for screwing a threaded sample holding device on and off a positioning device. The '849 patent uses a multi-axis robot to move crystals from a sample rack to a positioning device.